Use of autologous sediment from fluid aspirates as vehicles for drug delivery

ABSTRACT

A method of using fluid aspirates as vehicles for drug delivery by collecting a fluid aspirate such as synovial joint effusion, pleural effusion, pericardial effusion, or ascites from a patient and centrifuging the fluid aspirate to provide a supernatant and a sedimented material. The sedimented material can optionally be further purified. One or more factors such as cytokines, bone morphogenetic proteins (BMPs), pharmaceutical drugs and gene vectors are added to the sedimented material or supernatant, optionally also including a biologically compatible medium such as a bioabsorbable sponge, so as to provide a vehicle for the one or more factors for reintroducion into the patient.

PRIORITY

The present application claims priority under 35 USC section 119 andprovisional application Ser. No. 60/716, 064 filed on Sep. 12, 2005.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates generally to vehicles for delivering oneor more factors such as cytokines, bone morphogenetic proteins (BMPs),pharmaceutical drugs and gene vectors. Specifically, the presentinvention relates to the use of autologous sediment from fluid aspiratesas delivery vehicles.

(2) Description of the Related Art

Solutions, suspensions and emulsions have been used throughout the yearsas vehicles for delivery of the active ingredients of pharmaceuticaldrugs. These delivery vehicles do not allow for the maintenance ofeffective dosage levels of the active ingredients in the bloodstream.Sustaining a dosage of a therapeutic factor may require multipleinjections, which can increase the likelihood of infection. Therapeuticfactors such as pharmaceutical drugs and recombinant proteins oftenrequire controlled and sustained release at specific target tissues tobe safe and effective. If there is a narrow difference betweentherapeutic and toxic levels (therapeutic index) of a drug it willrequire strict compliance to an injection schedule by the patient.Additionally, cytokines such as IL-2 have a danger of systemic toxicity.IL-2 has useful local therapeutic potential, but systemically it cancause vascular shock and pulmonary edema. Another concern is thattherapeutic peptides have a very short half-life, so that targeted,controlled and sustained release is important for their effectiveness.

Recent advancements in the field of delivery vehicles allow for thecontrolled and sustained delivery of drugs. The advancements includesuch technologies as osmotic pumps, liposomes, dendrimers, andmicroencapsulation in biodegradable polymers such as microparticles,microspheres or nanoparticles. U.S. Pat. No. 4,489,055 to Couvreur etal., for example, describes biodegradable particles ofalkyl-cyano-acrylate containing a biologically active substance.Particles comprised of various polymers and copolymers, such as PLG[poly(lactide-co-glycolide)], PCL [poly( ,-caprolactone)], PLA[poly(L-lactic acid)] and PBLA [poly(∃-benzyl-L-aspartate)] have beendescribed (M. Ravi Kumar J. Pharm. Parmaceut. Sci. 3(2): 234-258, 2000).Alginate (including calcium alginate beads encapsulated withpoly-L-lysine) and chitosan have both been used extensively to createmicrocapsules and microspheres. Maintaining a minimal inflammatoryresponse to the vehicle is important in any design for a deliveryvehicle that is to be placed within the human body. Other advancementswhich allow for targeted and controlled release of factors include genetherapy. The use of a patient's own cells to carry the factor avoidssome of the issues relating to immune rejection, since the drug vehicleis autologous.

One difficult tissue of the body to target with drugs or other factorsis the synovium. Some have described the use of synovial fluidconstituents for injection. For example, U.S. Pat. No. 4,141,973 toBalazs describe a purified high molecular weight hyaluronic acidfraction extracted from animal tissues for injection into a joint. U.S.Pat. No. 5,079,236 to Drizen et al. describe a purified high molecularweight hyaluronic acid fraction for treatment of joint disease inanimals. HYALGAN® sodium hyaluronate (Sanofi-Synthelabo Inc, New York,N.Y.) is a purified hyaluronate from rooster combs for injection intoknee joints for the purpose of pain relief. U.S. Pat. No. 6,699,471 B2and U.S. Patent Application Publication No. 2004/0142465 A1 to Radice etal. describe injectable compositions having hyaluronic acid derivativesand cells such as chondrocytes for the treatment of soft tissues.CARTICEL® autologous cultured chondrocytes (Genzyme, Cambridge, Mass.)are presently used for the repair of articular cartilage defects causedby acute or repetitive trauma. The therapeutic chondrocytes are derivedfrom an in vitro expansion of autologous chondrocytes harvested from thenormal, femoral articular cartilage of the patient to be treated. Thecells are isolated and expanded, then implanted into the articularcartilage defect beneath an autologous periosteal flap sutured over thecartilage defect.

The synovium and synovial fluid in patients with rheumatoid arthritisare known to have upregulated proinflammatory cytokines.Antiinflammatory agents are activated in the disease, but do not counterthe proinflammatory response. Interferon-∃ (IFN-∃) is a naturalanti-inflammatory, because it downregulates proinflammatory cytokinessuch as IL-1∃ and tumor necrosis factor-∀ (TNF-∀) while also increasingthe IL-1 receptor antagonist in synoviocytes. Van Holten et al. (Arth.Res., vol. 6, no. 3) teach treatment in an animal model of rheumatoidarthritis using intraperitoneal injections of IFN-∃ to ameliorate thearthritis. However, this requires systemic treatment with the IFN-∃.Locally targeted therapy would be desirable. Bandara et al., Proc. Natl.Acad. Sci, USA, vol. 90, pp. 107641-10768 (1993) and Makarov et al.,Proc. Natl. Acad. Sci, USA, vol. 93, pp. 402-406 (1996) take anotherapproach by transducing synoviocytes with a cDNA so as to express theinterleukin 1 receptor-antagonist (IL-1ra) protein. Del Vecchio et al.(Arth. Res., vol. 3, no. 4) teach approaches to enhance the transductionof human synoviocytes with the interleukin 1 receptor-antagonist(IL-1ra) cDNA. The ex vivo transfer of genes for delivering genes to thesynovial lining of joints seems to selectively target type Bsynoviocytes. In vivo gene delivery by intra-articular injection ofadenovirus vectors apparently transduces leukocytes and both type A andB synoviocytes (Evans, Arth. Res., vol. 1 no. 1, pp. 21-24, 1999).Research by Ghivizzani et al. (Proc Natl Acad Sci, USA 1998,95:4613-4618) shows a contralateral effect of in vivo gene delivery,which suggests that transduced leukocytes have the capacity to trafficbetween joints.

While the related art teach various drug delivery vehicles which givecontrolled and sustained release, and while some related art utilizesynovial fluid constituents such as hyaluronic acid for the treatment ofjoint disease, there still exists a need for improved delivery vehiclesfor factors, such as drugs, gene vectors and cytokines which allow fortargeted, controlled and sustained release of the factors.

OBJECTS

Therefore, it is an object of the present invention to provide a meansto deliver one or more factors to a patient.

It is further an object of the present invention to provide a means todeliver of one or more factors to the patient utilizing autologousmaterial so as to minimize any inflammatory response.

These and other objects will become increasingly apparent by referenceto the following description.

SUMMARY OF THE INVENTION

The present invention provides a method of delivering one or morefactors to a patient which comprises collecting a fluid aspirate fromthe patient, centrifuging the fluid aspirate to provide a supernatantand a sedimented material, separating the supernatant from thesedimented material, immersing the sedimented material in a solutioncomprising one or more factors so as to provide a treated sediment, andintroducing the treated sediment to deliver the one or more factors tothe patient.

In further embodiments the fluid aspirate is synovial joint effusion,pleural effusion, pericardial effusion, or ascites. In still furtherembodiments the one or more factors are introduced to repair cartilagein a joint. In still further embodiments the one or more factors arecytokines, bone morphogenetic proteins (BMPs), pharmaceutical drugs,gene vectors or mixtures thereof. In still further embodiments thesedimented material after separating, and before immersing, is examinedand treated to remove unwanted components, to supply wanted componentsor both.

The present invention provides a method of delivering one or morefactors to a patient which comprises collecting a fluid aspirate fromthe patient, centrifuging the fluid aspirate to provide a supernatantand a sedimented material, separating the supernatant from thesedimented material, immersing the sedimented material in a solutioncomprising one or more factors, pressurizing the sedimented material inthe solution comprising one or more factors so as to provide a treatedsediment, and introducing the treated sediment to deliver the one ormore factors to the patient.

In further embodiments the fluid aspirate is synovial joint effusion,pleural effusion, pericardial effusion, or ascites. In still furtherembodiments the one or more factors are introduced to repair cartilagein a joint. In still further embodiments the one or more factors arecytokines, bone morphogenetic proteins (BMPs), pharmaceutical drugs,gene vectors or mixtures thereof. In still further embodiments thesedimented material after separating, and before immersing, is examinedand treated to remove unwanted components, to supply wanted componentsor both.

The present invention provides a method of delivering one or morefactors to a patient which comprises collecting a fluid aspirate fromthe patient, centrifuging the collected fluid aspirate to provide asupernatant and sedimented material, separating the supernatant from thesedimented material, immersing the sedimented material in a solutioncomprising one or more factors to provide a treated sediment, placingthe treated sediment into a biologically compatible medium, andintroducing the treated sediment and biologically compatible medium intoa tissue of the patient so as to deliver the one or more factors to thepatient.

In further embodiments the biologically compatible medium is blood or afibrin blood clot. In still further embodiments the biologicallycompatible medium is a bioabsorbable sponge. In still furtherembodiments the fluid aspirate is synovial joint effusion, pleuraleffusion, pericardial effusion, or ascites. In still further embodimentsthe one or more factors are introduced to repair cartilage in a joint.In still further embodiments the one or more factors are cytokines, bonemorphogenetic proteins (BMPs), pharmaceutical drugs, gene vectors ormixtures thereof. In still further embodiments the sedimented materialafter separating, and before immersing, is examined and treated toremove unwanted components, to supply wanted components or both.

The present invention provides a method of delivering one or morefactors to a patient which comprises collecting a fluid aspirate fromthe patient, centrifuging the collected fluid aspirate to provide asupernatant and sedimented material, separating the supernatant from thesedimented material, immersing the sedimented material in a solutioncomprising one or more factors, pressurizing the sedimented material inthe solution comprising one or more factors so as to provide a treatedsediment, placing the treated sediment into a biologically compatiblemedium, and introducing the treated sediment and biologically compatiblemedium into a tissue of the patient so as to deliver the one or morefactors to the patient.

In further embodiments the biologically compatible medium is blood or afibrin blood clot. In still further embodiments the biologicallycompatible medium is a bioabsorbable sponge. In still furtherembodiments the fluid aspirate is synovial joint effusion, pleuraleffusion, pericardial effusion, or ascites. In still further embodimentsthe one or more factors are introduced to repair cartilage in a joint.In still further embodiments the one or more factors are cytokines, bonemorphogenetic proteins (BMPs), pharmaceutical drugs, gene vectors ormixtures thereof. In still further embodiments the sedimented materialafter separating, and before immersing, is examined and treated toremove unwanted components, to supply wanted components or both.

The present invention provides a method of delivering one or morefactors to a patient which comprises collecting a fluid aspirate fromthe patient, centrifuging the fluid aspirate to provide a supernatantand a sedimented material, separating the supernatant from thesedimented material, purifying a one or more components of thesedimented material, immersing the one or more components in a solutioncomprising one or more factors so as to provide a treated vehicle, andintroducing the treated vehicle to deliver the one or more factors tothe patient.

In further embodiments the fluid aspirate is synovial joint effusion,pleural effusion, pericardial effusion, or ascites. In still furtherembodiments the one or more factors are introduced to repair cartilagein a joint. In still further embodiments the one or more factors arecytokines, bone morphogenetic proteins (BMPs), pharmaceutical drugs,gene vectors or mixtures thereof. In still further embodiments thepurified components of the purification step are examined to determinethe purity of the components prior to immersing.

The present invention provides a method of delivering one or morefactors to a patient which comprises collecting a fluid aspirate fromthe patient, centrifuging the fluid aspirate to provide a supernatantand a sedimented material, separating the supernatant from thesedimented material, purifying a one or more components of thesedimented material, immersing the one or more components in a solutioncomprising one or more factors, pressurizing the one or more componentsin the solution comprising one or more factors so as to provide atreated vehicle, and introducing the treated vehicle to deliver the oneor more factors to the patient.

In further embodiments the fluid aspirate is synovial joint effusion,pleural effusion, pericardial effusion, or ascites. In still furtherembodiments the one or more factors are introduced to repair cartilagein a joint. In still further embodiments the one or more factors arecytokines, bone morphogenetic proteins (BMPs), pharmaceutical drugs,gene vectors or mixtures thereof. In still further embodiments thetreated vehicle is examined in the step of pressurizing, before beingintroduced into the patient.

The present invention provides a method of delivering one or morefactors to a patient which comprises collecting a fluid aspirate fromthe patient, centrifuging the collected fluid aspirate to provide asupernatant and sedimented material, separating the supernatant from thesedimented material, purifying a one or more components of thesedimented material, immersing the one or more components in a solutioncomprising one or more factors, placing the one or more components inthe solution comprising one or more factors into a biologicallycompatible medium so as to provide a treated vehicle, and introducingthe treated vehicle into a tissue of the patient so as to deliver theone or more factors to the patient.

In further embodiments the biologically compatible medium is blood or afibrin blood clot. In still further embodiments the biologicallycompatible medium is a bioabsorbable sponge. In still furtherembodiments the fluid aspirate is synovial joint effusion, pleuraleffusion, pericardial effusion, or ascites. In still further embodimentsthe one or more factors are introduced to repair cartilage in a joint.In still further embodiments the one or more factors are cytokines, bonemorphogenetic proteins (BMPs), pharmaceutical drugs, gene vectors ormixtures thereof. In still further embodiments the purified componentsof the purifying step are examined to determine the purity of thecomponents prior to the immersing step.

The present invention provides a method of delivering one or morefactors to a patient which comprises collecting a fluid aspirate fromthe patient, centrifuging the collected fluid aspirate to provide asupernatant and sedimented material, separating the supernatant from thesedimented material, purifying a one or more components of thesedimented material, immersing the one or more components in a solutioncomprising one or more factors, pressurizing the one or more componentsin the solution comprising one or more factors, placing the one or morecomponents in the solution comprising one or more factors into abiologically compatible medium so as to provide a treated vehicle, andintroducing the treated vehicle into a tissue of the patient so as todeliver the one or more factors to the patient. In still furtherembodiments the purified components of the purifying step are examinedto determine the purity of the components prior to the immersing step.

In further embodiments the biologically compatible medium is blood or afibrin blood clot. In still further embodiments the biologicallycompatible medium is a bioabsorbable sponge. In still furtherembodiments the fluid aspirate is synovial joint effusion, pleuraleffusion, pericardial effusion, or ascites. In still further embodimentsthe one or more factors are introduced to repair cartilage in a joint.In still further embodiments the one or more factors are cytokines, bonemorphogenetic proteins (BMPs), pharmaceutical drugs, gene vectors ormixtures thereof.

The present invention provides a method of delivering one or morefactors to a patient which comprises: (a) collecting a fluid aspiratefrom the patient; (b) centrifuging the fluid aspirate to provide asupernatant and a sedimented material; and (c) separating thesupernatant from the sedimented material; (d) providing one or morefactors to the supernatant so as to provide a mixture; and (e) injectingthe mixture into the patient to deliver the one or more factors to thepatient. In further embodiments the fluid aspirate is synovial jointeffusion, pleural effusion, pericardial effusion, or ascites. In stillfurther embodiments the one or more factors are injected to repaircartilage in a joint. In still further embodiments the one or morefactors are a cytokine. In still further embodiments the one or morefactors are bone morphogenetic proteins (BMPs). In still furtherembodiments the one or more factors are a pharmaceutical drug. In stillfurther embodiments the one or more factors are a gene vector.

The present invention provides a method of delivering one or morefactors to a patient which comprises: (a) collecting a fluid aspiratefrom the patient; (b) centrifuging the collected fluid aspirate toprovide a supernatant and sedimented material; and (c) separating thesupernatant from the sedimented material; (d) placing a biologicallycompatible medium into the supernatant; (e) providing one or morefactors to the supernatant and biologically compatible medium so as toprovide a therapeutic mixture; and (f) placing the therapeutic mixtureinto a tissue of the patient so as to deliver the one or more factors tothe patient.

In further embodiments the biologically compatible medium is blood or afibrin blood clot. In still further embodiments the biologicallycompatible medium is a bioabsorbable sponge. In still furtherembodiments the fluid aspirate is synovial joint effusion, pleuraleffusion, pericardial effusion, or ascites. In still further embodimentsthe one or more factors are to repair cartilage in a joint. In stillfurther embodiments the one or more factors are cytokines, bonemorphogenetic proteins (BMPs), pharmaceutical drugs, or gene vectors. Instill further embodiments the supernatant after step (c) is examined andtreated to remove unwanted components.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a side view of a centrifuge tube (10) having a collection tube(20) that doubles as pressure chamber and a delivery syringe and homefor the drug or drug combination which can be used to perform the methodof the present invention.

DETAILED DESCRIPTION OF THE INVENTION

All patents, patent applications, government publications, governmentregulations, and literature references cited in this specification arehereby incorporated herein by reference in their entirety. In case ofconflict, the present description, including definitions, will control.

Synovium constitutes the lining of synovial joints. It consists ofseries of cells covering lining of fat and vascularity. The cellssecrete synovial fluid. These cells naturally shed and can be found insmall numbers in synovial fluid. In joint inflammation the liningproliferates into fingerlike projections called villi. These finger likeprojections are lined with synovial cells and filled with fat andvessels. Therefore there are synovial cells, fat cells with potentialfor some stem cells, fibroblasts, blood with monocytes and lymphocytesplus angioblasts. The latter are there related to the reaction of thesynovium and the increased vascularity. It has been reported by Hunzikerand Rosenberg that synovium will grow over cartilage and heal alaceration in cartilage (J Bone Joint Surg Am. 1996 May; 78(5):721-33).

Body fluids such as synovial fluid contain a variety of materials thatwhen isolated can serve as vehicles for drug and gene delivery. Forexample the synovial joint effusion that accompanies degenerativearthritis has a variety of debris. The fluid can be removed byarthrocentesis. The fluid contains cellular and tissue debris that isoften visible to the naked eye. When subjected to centrifuging, thematerial is separated out and collectable from the centrifuge tube.(Johnson, L L. Arthroscopic Surgery Principles and Practice. C. V. Mosby1986, St. Louis). When joint fluid undergoes centrifugation the sedimenthas components including, but not limited to white blood cells, redblood cells, synovial cells, synovial fragments, and articular cartilagefragments with and without viable appearing cells.

Protocol: One embodiment of the method involves the separation of theautologous joint fluid tissue debris by centrifuging and discarding thesupernatant. The sediment from the centrifugation is saved. Optionally,blood or a fibrin blood clot can be added. The sediment is immersed inone or more factors, for example a drug or gene vector, for up to 30minutes. The one or more factors is adsorbed over various times onto thevarious components which make up the sediment. Actuation of pressure onthe debris and the factors is one means encompassed by the presentinvention to increase the saturation of the drug or other factors in thedebris. The autologous sediment with adsorbed drug or gene vector isthen injected into the patient for the intended purpose. The drug orgene vector is selectively released from each constituent of thesediment at a different rate, according to cell and tissue type, givinga prolonged and even timed release of the drug. In one embodiment shownin FIG. 1, a sterile, disposable centrifuge tube (10) is used forperforming the methods of the present invention which can be used duringoutpatient surgery, or in a hospital surgery operating theater. Thecentrifuge tube (10) apparatus has a collection tube (20) that doublesas pressure chamber and a delivery syringe and home for the drug or drugcombination. In one example, the centrifuge tube (10) apparatuscomprises a collection tube (20) that doubles as a delivery syringewhich is inverted within a holder (30) during centrifugation. Thecollection tube (20) rests upon ledges (31) in the holder (30) so that aplunger (21) remains towards an open end (22) of the collection tube(20) during centrifugation. The collection tube (10) can be removed fromthe holder (30) after separation of the sediment from the fluid. Thesupernatant can then be removed from the collection tube (20) bypressing the handle (23). The remaining sediment can then be resuspendedby shaking or vortexing. Another example of a centrifugation syringewhich can be utilized to perform the method of the present invention isdisclosed in U.S. Pat. No. 5,577,513 to Van Vlasselaer herebyincorporated herein by reference in its entirety. The deliveryinstrument could be as simple as a syringe and needle. The materialcould be delivered in a autogenous fibrin blood clot, via abioabsorbable sponge, or injected under a patch of autogenous tissue.

One example of this is the treatment of cartilage injury or disease. Theinjured or degenerative joint has fluid with cells, cell debris,synovium, synovial cells, cartilage matrix, cartilage with matrix andcells. A cytokine such as one of the Bone Morphogenetic Proteins (BMPs)is mixed with sediment. The combination is then placed into the jointwith or without a medium such as a bioabsorbable sponge. BMPs areproteins within the transforming growth factor- beta (TGF-∃) superfamilywhich bind to serine/threonine transmembrane receptors thatphosphorylate Smad second messenger family proteins which regulatetranscription of various genes. A subfamily of BMPs, called GDFs, arelocalized in joints during development and therefore may be critical forsynovial joint morphogenesis. The BMPs, among other growth factors, canbe delivered directly as a protein or via gene vectors. Other examplesof sediments from fluid aspirates which can be used to provide vehiclesfor delivery of factors such as drugs and genes are those obtained frompleural effusion, pericardial effusion and ascites.

In another embodiment, the supernatant fluid remaining aftercentrifugation is utilized. In this embodiment, the particles would beremoved and only the lubricant proteins would remain in the synovialfluid. Cartilage debris is thereby removed. The proteins which are inthe supernatant are analyzed, and then mixed with one or more factors,for example BMP, and reinjected into the patient. A disposablecentrifuge tube (10) such as described previously is used. The syringecan be already coated with one or more factors, such as BMP, whenaspirating the surface synovial fluid in the centrifuge tube (10). Thecontents of the syringe are then injected at a certain time interval. Insome embodiments the contents are injected immediately.

Optionally, in some embodiments, the precipitated tissues are examinedfor diagnostic purposes prior to use. Some materials which have beencollected may be detrimental to the patient and these unwantedcomponents must be removed, while other materials may be helpful toreintroduce into a patient. For example, certain proteins and orcellular debris may cause an immune response or inflammation in thepatient. In some embodiments which utilize the supernatant forintroduction into the patient, specific proteins or all proteinaceousmaterial can be extracted or bound before the patient receives thesupernatant materials. For diagnostic analysis, the materials can becentrifuged and the precipitates and smears of the supernatant can beexamined morphologically and histochemically for their nature andacceptability for purity and subsequent use. The precipitant can beexamined including placement in paraffin blocks for histologicalanalysis.

EXAMPLE

A synovial joint fluid aspirate is to be collected from a knee joint ofa patient. The fluid aspirate is then centrifuged to provide asupernatant and a sedimented material. The supernatant is then beremoved from the sedimented material and one or more factors such ascytokines and bone morphogenetic proteins (BMPs) are then provided tothe supernatant so as to provide a therapeutic mixture. Prior toinjecting the mixture into the patient to deliver these factors, themixture can be tested on alternate knees in a laboratory animal todetermine whether the prepared therapeutic mixture is sufficientlyclean. Treated versus untreated knees of the laboratory animal can bethen compared. If it is determined that the mixture is sufficientlyclean, the therapeutic mixture can be then be injected into the knee ofthe patient which requires treatment.

While the present invention is described herein with reference toillustrated embodiments, it should be understood that the invention isnot limited hereto. Those having ordinary skill in the art and access tothe teachings herein will recognize additional modifications andembodiments within the scope thereof. Therefore, the present inventionis limited only by the Claims attached herein.

1. A method of delivering one or more factors to an injured ordegenerative joint of a patient which comprises: (a) collecting a fluidaspirate from the injured or degenerative joint of the patient, whereinthe injured or degenerative joint comprises synovial villi; (b)centrifuging the fluid aspirate to provide a supernatant and asedimented material, wherein the sedimented material comprises cellularcomponents; (c) separating the supernatant from the sedimented material;(d) immersing the sedimented material in a solution comprising one ormore factors to stimulate cartilage growth so as to provide a treatedsediment; and (e) introducing the treated sediment to deliver the one ormore factors to the joint of the patient, wherein the method isperformed with a centrifuge tube apparatus comprising a syringe and aholder, wherein the syringe comprises a collection tube comprising anend with a narrowed outlet and a means for connection to a needle and anopen end, wherein the syringe further comprises a plunger inserted intothe open end, wherein steps (a), (c), (d) and (e) are performed usingthe syringe, and wherein during step (b) the syringe rests in aninverted position upon ledges in the holder so that the plunger remainstowards the open end of the syringe.
 2. The method of claim 1 whereinthe sedimented material after step (c), and before step (d), is examinedand treated to remove unwanted components, to supply wanted componentsor both.
 3. The method of claim 1 wherein the fluid aspirate is synovialjoint effusion, pleural effusion, pericardial effusion, or ascites. 4.The method of claim 1 wherein the one or more factors are introduced torepair cartilage in a joint.
 5. The method of claim 1 wherein the stepof immersing the sedimented material in the solution further comprisespressurizing the sedimented material in the solution.
 6. The method ofclaim 1 further comprising a step of placing the treated sediment into abiologically compatible medium, further wherein the introduced treatedsediment is introduced together with the biologically compatible medium.7. The method of claim 6 wherein the biologically compatible medium isblood or a fibrin blood clot.
 8. The method of claim 6 wherein thebiologically compatible medium is a bioabsorbable sponge.
 9. The methodof claim 1 further comprising a step of purifying one or more sedimentcomponents from the sedimented material to form a purified sedimentedmaterial, further wherein the step of immersing sedimented material isimmersing purified sedimented material.
 10. The method of claim 9wherein the purity of the one or more sediment components is determinedfurther comprising determining the purity of the one or more sedimentedcomponents.
 11. The method of claim 9 further comprising a step ofplacing the treated sediment into a biologically compatible medium,wherein the introduced treated sediment is introduced together with thebiologically compatible medium.
 12. The method of claim 1 wherein thecellular components are synovial cells or synovial fragments.
 13. Themethod of claim 1 wherein the cellular components are articularcartilage fragments.
 14. The method of claim 1 wherein the cellularcomponents are fibroblasts or angioblasts.
 15. The method of claim 1wherein the cellular components comprise stem cells.
 16. The method ofclaim 1 wherein the one or more factors to stimulate cartilage growthare selected from the group consisting of a cytokine, a bonemorphogenetic protein (BMP) and a BMP encoding nucleic acid molecule ina nucleic acid vector.
 17. The method of claim 1 wherein the treatedsediment is introduced to intra-articular structures selected from thegroup consisting of a meniscus, articular cartilage, a ligament.
 18. Themethod of claim 17 wherein the sediment is introduced duringreconconstruction or grafting.